Removal of bacterial endotoxin in a protein solution by immobilized metal affinity chromatography

ABSTRACT

The present invention relates to the purification of polypeptides and the removal of endotoxin via immobilized metal affinity chromatography (IMAC). More specifically, the invention relates to methods for removing bacterial endotoxin in a protein solution. In specific embodiments, the invention relates to the elimination of endotoxin from  Moraxella catarrhalis  outer membrane proteins.

This application is a divisional of U.S. application Ser. No.10/474,533, filed Oct. 10, 2003, now U.S. Pat. No. 6,942,802, which is afiling under 35 USC 371 of PCT/US02/10937 filed Apr. 5, 2002, whichclaims the benefit of provisional application Ser. No. 60/283,728, filedApr. 13, 2001, the entire disclosure of each application is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to the fields of bacteriology,toxicology and protein purification. In particular, the inventionrelates to the purification of polypeptides and the removal of endotoxinvia immobilized metal affinity chromatography (IMAC). More specifically,the invention relates to methods for removing bacterial endotoxin in aprotein solution. In specific embodiments, the invention relates to theelimination of endotoxin from Moraxella catarrhalis outer membraneproteins.

BACKGROUND OF THE INVENTION

Despite aggressive management, septic shock arising from Gram-negativebacteria sepsis continues to be a leading cause of death in bothsurgical and medical patients. Death in such patients usually resultsfrom cardiovascular collapse and/or multiple organ system failure. Oneof the most numerous and dominant agents causing sepsis fromGram-negative bacterial infection is endotoxin, which is present on thesurface of Gram-negative bacteria, including Escherichia coli.

Bacterial endotoxin is a complex consisting of lipid, carbohydrate andprotein. It is characterized by an overall negative charge, heatstability and high molecular weight. Highly purified endotoxin does notcontain protein, and is comprised of lipopolysaccharide (LPS) andlipooligosaccharide (LOS). Depyrogenation can generally be achieved byinactivating or removing endotoxin, depending upon the physicochemicalnature of the LPS. LPS consists of three distinct chemical regions,lipid A, which is the innermost region, an intermediate corepolysaccharide, and an outermost O-specific polysaccharide side chain,which is responsible for an endotoxin's particular immunospecificity.

Bacterial endotoxins present severe pathophysiological reactions whenintroduced into animals, including high fever, vasodilation, diarrheaand, in extreme cases, fatal shock. (Morrison, 1987). Thus, it iscritical to avoid endotoxin contamination in any pharmaceutical productor medical device which comes into contact with body fluids. Inaddition, high endotoxin levels in sera due to bacterial diseases, suchas septicemia, are not easily treated. Antibiotic treatment of theinfection only kills the bacteria, leaving the endotoxin from their cellwalls free to cause fever.

Endotoxins tend to form micellar structures which have a similardensity, size, and charge distribution on the outer surface of themicelles. As a result, endotoxins co-purify with proteins or nucleicacids. Various attempts have been made to eliminate endotoxins presentin biological and pharmaceutical compositions (U.S. Pat. Nos. 5,972,225;6,132,610; 6,194,562; 5,747,455; 5,169,535; 5,101,019; 4,808,314;4,059,512 and 3,959,128) and it has become increasingly evident, thatendotoxins are not readily separated from protein or nucleic acidsamples. Further, endotoxins are extremely stable, resist extremes oftemperature and pH value and have a broad spectrum of biologicalactivity (e.g., are toxic in humans and other animals, are pyrogenicwhen present in trace amounts, and can cause hypotensive shock,disseminated intravascular coagulation and death).

Bacterial endotoxins thus impede progress in various areas ofbiotechnology. Gram-negative bacteria can shed endotoxins from theircell walls and endotoxins are therefore a potential contaminant of anyaqueous solution. For example, during lysis of bacterial cells, such asis done in recombinant protein purification or to release plasmids fromtransformants (e.g., E. coli), endotoxins are released into the lysateproduced thereby. Endotoxin contamination in protein or nucleic acidsamples can adversely limit the utility of the sample, particularly inapplications which are sensitive to such contamination (e.g.,pharmaceutical compositions). For example, the transfection efficienciesof several different cultured eukaryotic cell lines, including HeLa,Huh7, COS7, and LMH, have been shown to be sharply reduced in thepresence of endotoxins (Weber et al., 1995). Endotoxins have also beenfound to be toxic to primary human cells, such as primary human skinfibroblasts and primary human melanoma cells, in the presence ofentry-competent adenovirus particles (Cotton et al., 1994).

Although glassware, plasticware, water, and most buffers can beeffectively decontaminated from free endotoxins (see for example, Sofer,1984; Issekutz, 1983), many proteinaceous macromolecules such ashormones, immunoglobulins, and enzymes are biologically inactivefollowing such treatments. This is a particularly important problem withthe recent advances in biotechnology. Bacterial contamination of usefulbiological products is recognized as a problem (Wightsmith et al.,1982). Endotoxin-producing bacteria used in genetic engineeringexperiments can add greatly to the risk of endotoxin contamination ofmaterials produced by such techniques.

Ultrafiltration, dialysis and certain chromatographic methods have beenemployed to remove endotoxin from aqueous solutions. These methodstypically separate small molecules from endotoxins based on the sizedifference between the small molecule and endotoxin, which aggregatesinto high molecular weight micelles in aqueous solutions. However,endotoxins and many macromolecules are often too similar in size to beseparated using these techniques. Additional chromatographicpurification techniques, such as adsorbing matrices, affinitychromatography and ion exchange chromatography, have been described toremove endotoxin in a solution (U.S. Pat. Nos. 3,897,309; 4,276,050;4,381,239; Morrison et al., 1976; Duff et al., 1982; Issekutz, 1983).However, these procedures typically still permit about 10% of theoriginally present endotoxin to remain in solution or associated withprotein following elution from the column (e.g., see Duff et al., 1982).The presence of that 10% protein-associated endotoxin may not affect theendotoxin assay, but still could remain pyrogenic.

Moraxella catarrhalis is an important human respiratory tract pathogen.M. catarrhalis is the third most common cause of otitis media in infantsand children (Murphy, 1989). Moraxella catarrhalis is a common cause ofsinusitis and conjunctivitis in both children and adults (see e.g.,Bluestone, 1986; Brorson et al., 1976; Romberger et al., 1987) and is animportant cause of lower respiratory tract infections in adults withchronic bronchitis and chronic obstructive pulmonary disease (Murphy etal., 1992; Catlin, 1990). Additionally, M. catarrhalis can causepneumonia, endocarditis, septicemia, and meningitis in immunocomprisedhosts (Cocchi et al., 1968; Douer et al., 1977; McNeely et al., 1976).

Since recurrent otitis media is associated with substantial morbidity,and the attendant health care costs, there is interest in developingstrategies for identifying and preventing these infections. One suchapproach is the development of immunogenic compositions for preventingbacterial otitis media. Outer membrane proteins are being investigatedas antigens having utility in diagnosing and immunizing against diseasecaused by bacterial pathogens, such as M. catarrhalis. However, it isimperative in the formulation of these compositions, that the outermembrane protein antigen(s) is effectively free of bacterial endotoxin,so as to prevent sepsis.

Thus, there is presently a need for simple and efficient methods orprocesses to purify protein samples, effectively free of bacterialendotoxin. It is additionally desirable that such a protein purificationmethod occurs without significant loss in protein concentration orbiological activity, such that the protein can be administered (e.g.,parenterally) as a pharmaceutical or immunogenic composition,effectively free of endotoxin.

SUMMARY OF THE INVENTION

The present invention relates to the purification of polypeptides andthe removal of endotoxin by immobilized metal affinity chromatography(IMAC). More specifically, the invention relates to methods for removingbacterial endotoxin in a protein solution, and in specific embodiments,the elimination of endotoxin from Moraxella catarrhalis outer membraneproteins.

Thus, in particular embodiments the present invention is directed to amethod for removing bacterial endotoxin in a protein solution usingimmobilized metal ion affinity chromatography (IMAC) comprising thesteps of (a) applying the solution to an IMAC resin, wherein the resinis equilibrated in a buffer; (b) eluting the endotoxin with the bufferof step (a), wherein the protein remains bound to the resin; (c) elutingthe protein in an elution buffer comprising glycine; and (d) collectingthe eluted protein, wherein the concentration of the endotoxin in theprotein solution is effectively reduced by a factor of at least 200 andprotein recovery is greater than at least 60%. In preferred embodiments,the concentration of the endotoxin in the protein solution iseffectively reduced by a factor of at least 300, more preferably afactor of at least 500, even more preferably a factor of at least 1000and most preferably a factor of at least 2,500 and protein recovery isgreater than at least 65%, more preferably greater than at least 70%,even more preferably greater than at least 80%, yet more preferablygreater than at least 85%, and most preferably greater than at least 90to about 99.9%.

In particularly preferred embodiments, the endotoxin is alipooligosaccharide (LOS) or a lipopolysaccharide (LPS) and the proteinis a Gram negative bacteria protein. In a preferred embodiment, the Gramnegative bacteria protein is a Moraxella catarrhalis outer membraneprotein, more preferably, a Moraxella catarrhalis UspA2 protein. Inother embodiments of the invention, the buffer of step (a) is selectedfrom the group consisting of sodium phosphate, sodium acetate,1,4-piperazinebis-(ethanesulfonic acid) (PIPES),N-(2-acetamido)imino-diacetic acid (ADA) andN-(2acetamido)-2-aminoethanesulfonic acid (ACES) and the elution buffercomprising glycine of step (c) is selected from the group consisting ofsodium phosphate, sodium acetate, imidazole, histidine, ammoniumchloride, PIPES, ADA and ACES. In particular embodiments, the elutionbuffer of step (c) has a glycine concentration from about 1 mM to about500 mM. In a preferred embodiment, the buffer of step (a) is sodiumphosphate at a concentration of at least about 1 mM to about 50 mM andhas a pH of about 6.5 to about 7.8, the buffer further comprising about150 mM NaCl and 0.1% Triton-X. In a particularly preferred embodiment,the sodium phosphate has a concentration of about 10 mM and a pH of 6.8.

In another preferred embodiment, the elution buffer of step (c) issodium phosphate at a concentration of at least about 1 mM to about 50mM and has a pH of about 6.5 to about 7.5, the buffer further comprisingabout 150 mM NaCl, 0.04% Triton-X and 100 mM glycine. In a particularlypreferred embodiment, the sodium phosphate has a concentration of about10 mM and a pH of 6.8.

In yet other embodiments of the invention, the IMAC resin is chargedwith a divalent or trivalent cation such as Cu²⁺, Ni²⁺, Fe³⁺, Co²⁺,Cd²⁺, Ti²⁺, Mg²⁺, Fe²⁺, Mn²⁺ or Zn²⁺. In preferred embodiments, theresin is charged with Cu²⁺, Ni²⁺, Zn²⁺ or Fe³⁺, more preferably Cu²⁺. Ina most preferred embodiment, the resin is charged with 20 mM cupricsulfate.

In another embodiment, the buffer of step (a) and the elution buffer ofstep (c) further comprise a mobile phase modifier selected from thegroup consisting of urea, ethanol, methanol, isopropanol, ethyleneglycol and a detergent.

In certain embodiments, the resin is washed prior to step (a) with atleast 3 to 10 resin volumes of the buffer used in step (c) and thenwashed with 1 to 10 resin volumes of the buffer of step (a) wherein thewashes remove excess copper from the resin. In another embodiment, theprotein solution is diluted in a buffer comprising 10 mM sodiumphosphate, 150 mM NaCl and 0.25% Triton-X at pH 6.9, before proceedingto step (a). In additional embodiments, the protein is further purifiedby ultrafiltration or filtration.

In a particularly preferred embodiment of the invention, an isolated andpurified UspA2 protein of Moraxella catarrhalis is provided, comprisingless than about 0.1 endotoxin units per ug of UspA2 protein. The UspA2protein is purified by IMAC, the method comprising the steps of (a)applying a Usp2A protein solution to an IMAC resin, wherein the resin isequilibrated in a buffer; (b) eluting the endotoxin with the buffer ofstep (a), wherein the protein remains bound to the resin; (c) elutingthe protein in an elution buffer; and (d) collecting the eluted protein.

Other features and advantages of the invention will be apparent from thefollowing detailed description, from the preferred embodiments thereof,and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention addresses the need for methods for treatingsolutions of proteins containing endotoxins to effectively reduce theconcentration of endotoxins contained therein. The invention describedherein offers a rapid and efficient means for removing endotoxins fromsuch solutions, thereby providing purified proteins which can be used ina variety of biological applications, including but not limited to, invivo administration of the purified proteins as immunogenic orpharmaceutical compositions.

Thus, in particular embodiments, the invention is directed to thepurification of polypeptides and the removal of endotoxin by immobilizedmetal affinity chromatography (IMAC). More specifically, the inventionrelates to methods for removing bacterial endotoxin in a proteinsolution, particularly, endotoxin from Moraxella catarrhalis outermembrane proteins by IMAC.

A. Endotoxin Removal and Protein Purification

The present invention is thus directed in particular embodiments, to theremoval of bacterial endotoxin. In a preferred embodiment, the inventionis directed to a method for removing bacterial endotoxin in a proteinsolution using immobilized metal ion affinity chromatography (MAC); themethod comprising the steps of applying the solution to an IMAC resin,eluting the endotoxin from the IMAC resin, wherein the protein remainsbound to the resin, eluting the protein in an elution buffer comprisingglycine and collecting the eluted protein, wherein the concentration ofthe endotoxin in the protein solution is effectively reduced by a factorof at least 200 and protein recovery is greater than at least 60%.

The use of the phrase “effectively reduced by a factor of at least 200,”refers to the endotoxin concentration as determined before and afterIMAC purification. Similarly, protein recovery according to the presentinvention, is the protein concentration as determined before and afterIMAC purification. In a preferred embodiment, the concentration of theendotoxin in the protein solution is effectively reduced by a factor ofat least 250, more preferably reduced by a factor of at least 500, evenmore preferably reduced by a factor of at least 1000 fold and mostpreferably reduced by a factor of at least 2000 fold. In anotherpreferred embodiment, protein recovery is greater than at least 65%,more preferably protein recovery is greater than at least 70%, morepreferably yet protein recovery is greater than at least 80%, and mostpreferably protein recovery is greater than about 95%. In a preferredembodiment, the invention is directed to an isolated and purified UspA2protein of Moraxella catarrhalis comprising less than about 0.1endotoxin units per ug of UspA2 protein, wherein the protein is purifiedby the above method.

As mentioned supra, the present invention relates to a method forremoving bacterial endotoxin in a protein solution using immobilizedmetal ion affinity chromatography (IMAC). The principles of IMAC aregenerally appreciated by those of skill in the art. It is believed thatadsorption is predicated on the formation of a metal coordinationcomplex between a metal ion, immobilized by chelation on the adsorbentmatrix, and accessible electron donor amino acids on the surface of theprotein to be bound. The metal ion microenvironment including, but notlimited to, the matrix, the spacer arm, if any, the chelating ligand,the metal ion, the properties of the surrounding liquid medium and thedissolved solute species can be manipulated by the skilled artisan toeffect the desired fractionation.

Not wishing to be bound by any particular theory as to mechanism, it isfurther believed that the more important amino acid residues in terms ofbinding are histidine, tryptophan and probably cysteine. Since one ormore of these residues are generally found in proteins, one might expectall proteins to bind to IMAC resins. However, the residues not only needto be present but also accessible (e.g. oriented on the surface of theprotein) for effective binding to occur to the resin. Thus, the presentinvention also contemplates the addition of appropriate residues to aprotein of interest. For example, poly-histidine tails added to theamino terminus or carboxy terminus of a protein, can be engineered intoa recombinant expression system (as described in U.S. Pat. No.4,569,794) to either permit or enhance resin binding. Thus, it iscontemplated here, that the addition of appropriate residues might beemployed in order to facilitate endotoxin removal from a proteinsolution, wherein the protein does not bind or binds weakly to the IMACresin in the absence of such residue additions.

The nature of the metal and the way it is coordinated on the resin canalso influence the strength and selectivity of the binding reaction.Matricies of silica gel, agarose and synthetic organic molecules such aspolyvinyl-methacrylate co-polymers can be employed. The matriciespreferably contain substituents to promote chelation. Substituents suchas iminodiacetic acid (IDA) or its tris (carboxymethyl) ethylene diamine(TED) can be used, wherein IDA is preferred. A particularly useful IMACmaterial is a polyvinyl methacrylate co-polymer substituted with IDAavailable commercially, e.g., as TOYOPEARL AF-CHELATE 650M (ToyoSodaCo.; Tokyo). Another useful IMAC ligand is nitrilotriacetic acid,comprising four available metal binding sites. The metals are preferablydivalent cations (e.g., Zn²⁺, Cu²⁺, Ni²⁺, Co²⁺, Fe²⁺, Mn^(2+, Ti) ²⁺,Cd²⁺ and Mg²⁺), with Cu²⁺ being the most preferred, but may also betrivalent cations such as Fe³⁺. An important selection parameter is ofcourse, the affinity of the protein to be purified for a particularmetal.

In practice the IMAC column is “charged” with a metal by pulsing with aconcentrated metal salt solution followed by water or buffer. A pre-washwith intended elution buffers is usually carried out. Sample buffers maycontain salt up to 1M or greater to minimize nonspecific ion-exchangeeffects. Adsorption of proteins is maximal at higher pHs. Elution isnormally either by lowering of pH to protonate the donor groups on theadsorbed protein, or by the use of stronger complexing agent such asimidazole, or glycine buffers. In these latter cases, the metal may alsobe displaced from the column. Linear gradient elution procedures canalso be beneficially employed.

Although the present invention is directed to the purification ofpolypeptides and the removal of endotoxin by immobilized metal affinitychromatography (IMAC), additional purification and/or processing of theprotein sample obtained by IMAC purification may additionally be carriedout using techniques common in the art. For example, the protein mayadditionally be purified by ion exchange chromatography, affinitychromatography, hydrophobic interaction chromatography, high purityliquid chromatography, fast purity liquid chromatography,ultrafiltration, diafiltration, dialysis, and/or ethanol precipitation.Further, the protein may be additionally concentrated byultrafiltration, diafiltration, ethanol precipitation and thebuffer/salt composition exchanged by dialysis, ultrafiltration, ethanolprecipitation and/or ion exchange chromatography.

Conditions such as salt concentrations, buffer composition, ionicstrength, pH and the like, are readily determined by the skilledartisan, and are often an empirical determination, dependent on themacromolecules to be separated or purified. For example, binding ofendotoxin according to the present invention typically occurs in the pHrange of 5.5-8.5. In addition, alternative buffers and concentrationranges for promoting endotoxin-IMAC binding are 0.01-0.2 M sodiumphosphate and 0.05 M sodium acetate. NaCl concentrations of 0.15-0.5 Mmay further be included in the “binding buffer” to prevent ion exchangeeffects. Commonly used buffers for elution are 0-0.5 M imidazole, 0-0.05M histidine, 0-2 M ammonium chloride. Chelating agents such as 0.05 MEDTA or EGTA may be used in certain formulations, but will strip thecolumn of the metal. Mobile phase modifiers such as urea, isopropanol(other alcohols), ethylene glycol or detergents may also be used toelute or improve separation (see, Franken et al., 2000).

The surface of Gram-negative bacteria comprises endotoxin, which can beshed endotoxins from their cell walls. Bacterial endotoxin is a complexconsisting of lipid, carbohydrate and protein. It is characterized by anoverall negative charge, heat stability and high molecular weight.Highly purified endotoxin does not contain protein, and is alipopolysaccharide (LPS). Depyrogenation can generally be achieved byinactivating or removing endotoxin, depending upon the physicochemicalnature of the LPS. LPS consists of three distinct chemical regions,lipid A, which is the innermost region, an intermediate corepolysaccharide, and an outermost O-specific polysaccharide side chain,which is responsible for an endotoxin's particular immunospecificity.Endotoxins tend to form micellar structures which have a similardensity, size, and charge distribution on the outer surface of themicelles. Some proteins, particularly lipoproteins and monoclonalantibodies, are known to disaggregate endotoxins and form complexes withLOS or LPS in solution, which increases the difficulty for separatingthe endotoxin and protein (Liping et al., 1997). Bacterial endotoxin istypically quantitated using the LAL Kinetic-Turbidimetric Method(Toxinometer Assay). In this method, the endotoxin concentration ismeasured by optically monitoring the increasing turbidity of a sample,resulting from an endotoxin-dependent gelation of the limulus amebocytelysate (LAL).

B. Recombinantly Expressed Moraxella Catarrhalis UspA2 Polypeptides

In certain embodiments of the invention, an isolated and purified UspA2outer membrane protein of Moraxella catarrhalis is provided, comprisingless than about 0.1 endotoxin units per ug of UspA2 protein. The UspA2protein is purified by IMAC, the method comprising the steps of (a)applying a UspA2 protein solution to an IMAC resin, wherein the resin isequilibrated in a buffer; (b) eluting the endotoxin with the buffer ofstep (a), wherein the protein remains bound to the resin; (c) elutingthe protein in an elution buffer comprising glycine; and (d) collectingthe eluted protein.

Thus, in particular embodiments, the invention contemplates thepurification of Moraxella catarrhalis UspA2 protein. Preferably, a UspA2protein of the invention is a recombinant protein. Typically, a UspA2protein is produced by recombinant expression in a non-human cell,preferably a prokaryotic cell, and most preferably a Gram-negative cell,in particular, an E. coli cell. In certain embodiments, a UspA2 proteinis a full length UspA2 protein, whereas in other embodiments it may be aUspA2 protein fragment. Expression vectors would thus comprise a UspA2polynucleotide or fragment thereof that encodes a UspA2 polypeptide orits fragment. Expression of proteins in prokaryotes is most oftencarried out in E. coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Fusion vectors add a number of amino acids to a proteinencoded therein, to the amino or carboxy terminus of the recombinantprotein.

Examples of inducible E. coli expression vectors include pTrc (Amann etal., 1988) and pET I I d (Studier et al., 1990). Vector DNA can beintroduced into prokaryotic or eukaryotic cells via conventionaltransformation, infection or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (“MolecularCloning: A Laboratory Manual” 2nd. Ed. Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

A host cell of the invention, such as a prokaryotic host cell inculture, can be used to produce (i.e., express) UspA2 polypeptides.Accordingly, the invention further provides methods for producing UspA2polypeptides using the host cells of the invention. The method comprisesculturing the host cell (into which a recombinant expression vectorencoding a UspA2 polypeptide has been introduced) in a suitable mediumuntil the UspA2 polypeptide is produced. The method further comprisesisolating the UspA2 polypeptide from the medium or the host cell.

In another aspect, the recombinant host cells of the present inventionare prokaryotic host cells. Preferably, the recombinant host cells ofthe invention are bacterial cells of the DH5 α strain of Escherichiacoli. In general, prokaryotes are preferred for the initial cloning ofDNA sequences and constructing the vectors useful in the invention. Forexample, E. coli K12 strains can be particularly useful. Other microbialstrains which can be used include E. coli B, and E. coli _(X)1976 (ATCCNo. 31537).

Other prokaryotic strains, such as E. coli W3110 (ATCC No. 273325),bacilli such as Bacillus subtilis, or other enterobacteriaceae such asSalmonella typhimurium or Serratia marcesans, and various Pseudomonasspecies can also be used for expression. These examples are, of course,intended to be illustrative rather than limiting.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli can betransformed using pBR322, a plasmid derived from an E. coli species(Bolivar, et al. 1977). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides easy means for identifyingtransformed cells. The pBR plasmid, or other microbial plasmid or phagemust also contain, or be modified to contain, promoters which can beused by the microbial organism for expression of its own polypeptides.

Those promoters most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase) and lactose promoter systems(Chang, et a? 1978; Itakura., et al. 1977, Goeddel, et al. 1979;Goeddel, et al. 1980) and a tryptophan (TRP) promoter system(International Application No. EP 0036776; Siebwenlist et al. 1980).While these are the most commonly used, other microbial promoters havebeen discovered and utilized, and details concerning their nucleotidesequences have been published, enabling a skilled worker to introducefunctional promoters into plasmid vectors (Siebwenlist, et al. 1980).

EXAMPLES

The following examples are carried out using standard techniques, whichare well known and routine to those of skill in the art, except whereotherwise described in detail. The following examples are presented forillustrative purpose, and should not be construed in any way limitingthe scope of this invention.

Example 1 Analytical Methods

SDS-PAGE AND LOS Western Blot Analysis

SDS-PAGE samples were prepared by mixing 1:1 with 2× sample buffer andheating at 100° C. for 10 minutes. Samples were loaded onto 4-12%(wt./vol.) Tris-glycine polyacrylamide gels (Zaxis) and stained withCoommassie blue (Zoion). LOS Western Blot analysis was performed using4-20% (wt./vol.) polyacrylamide gels that were transferred byelectrophoresis onto nitrocellulose membranes (BioRad). Thenitrocellulose was blocked in Tris buffered saline (TBS) containing 3%(wt./Vol.) BSA for 1 hour and then washed 3 times with Tris bufferedsaline containing 0.05% Tween 20 (TTBS). The membranes were placed in aprimary antibody solution containing a monoclonal antibody specific forlipooligosaccharide purified from Moraxella catarrhalis (MAb 73-11,lot#121294) diluted in TBS containing 1% BSA and incubated at roomtemperature for 1 hour. The membranes were washed 3 times with TTBSfollowed by incubation in the secondary antibody solution of Goatanti-mouse IgG-HRP (BioRad) diluted in TBS containing 1% BSA for 1 hourat room temperature. After incubation in the secondary antibodysolution, the membranes were washed 3 times with TTBS and then 3 timeswith TBS. The blots were developed using a substrate containing4-chloro-1-naphthol at a concentration of 1.4 g/l in an organic basemixed 1:1 with a solution composed of 0.02% H₂O₂ in a Citric Acid buffer(KPL, Gaithersburg, Md.) Moraxella cattarrahlis lipooligosaccharide(LOS) used as a standard was prepared from strain ATCC25238 by themodified Westphal method (Westphal and Jann, 1965).

Total Protein, Limulus Amebocyte Lysate (LAL) Assay and Residual CopperAnalysis

Total protein was determined by the microbicinchoninic acid assay(Pierce, Rockford, Ill.). LAL Analysis was performed according to theGuideline on Validation of the Limulus Amebocyte Lysate test as anEnd-product Endotoxin Test for Human and Animal Parenteral Drugs,Biological Products, and Medical Devices, December 1987, U.S. Departmentof Health and Human services, FDA and Interim Guidance for same, July1991. Residual copper analysis was performed by graphite Furnace atomicAbsorption Spectroscopy.

Example 2 Screening of UspA2 and LOS for Affinity to Chelated Metal Ions

UspA2 is an outer membrane protein purified from Moraxella catarrhalis,a Gram negative bacteria. The protein has a molecular mass of 62 kDa,but exists as an oligomer and runs at approximately 240 kDa by SDS-PAGEanalysis.

UspA2 and LOS were screened for affinity to chelated metal ions bound toIDA resin. Two milliliters of Pharmacia chelating sepharose fast flowresin was placed in a test tube. The resin was washed twice with 4 mL ofwater for injection (WFI), allowed to settle between washes and thesupernatant removed. The resin was then charged with 4 mL of one of thefollowing metal ion solutions: 5 mg/mL CuSO₄, 5 mg/mL NiSO₄, 5 mg/mLZnCL₂ or 0.1 M FeCl₃. After the resin settled, the supernatant wasremoved. This was followed by 5 washes of 4 mL WFI allowing the resin tosettle between washes and removal of the supernatant. The resin was thenwashed with 5-10 volumes of equilibration buffer (10 mM sodiumphosphate/0.5 M NaCl/0.1% Triton X-100/pH 7.2). A 2 mL solutioncontaining 0.1 mg/mL of purified UspA2 in equilibration buffer was addedto the resin. The resin was incubated in the solution for 15 minuteswith gentle shaking at room temperature. Then the resin was allowed tosettle and the supernatant was removed. The resin was washed with threevolumes of equilibration buffer, allowing the resin to settle andremoving the supernatant between washes. Four milliliters of elutionbuffer (10 mM sodium phosphate/0.5 M NaCl/100 mM glycine/0.1% TritonX-100/pH 7.0) was added to the resin and incubated with gentle shakingat room temperature. After incubation, the resin was allowed to settleand the supernatant removed. The resin was again treated with 4 mL ofelution buffer and incubated with gentle shaking at room temperature.After the second incubation, the resin was again allowed to settle andthe supernatant removed. The samples were pooled and analyzed bySDS-PAGE and LOS Western Blot.

SDS-PAGE analysis revealed that all of the UspA2 bound after loadingonto the Cu²⁺ charged resin and could not be detected in the supernatantThe protein bound less well to the Ni²⁺ charged resin with some residualprotein detected in the supernatant after loading. UspA2 binding to theZn²⁺ and Fe³⁺ charged resins was not detected. LOS Western Blot analysisindicated that the contaminating LOS in the UspA2 protein solution didnot bind to any of the metal ions tested. Virtually all of the LOSdetected by LOS Western Blot analysis was in the supernatant after theresin was loaded. Some residual LOS was detected in the supernatantafter elution of the protein, which is most likely due to residual LOSthat was not removed in the supernatant after loading. Based on theaffinity of UspA2 for Cu²⁺, the inventors proceeded to run a small scaleCu²⁺ charged IMAC column that would allow the purification of enoughprotein to test the final product for LAL.

Example 3 Small Scale Chromatography

A 157 mL (5 cm×8 cm) column was packed using Pharmacia ChelatingSepharose Fast flow resin according to the manufacturer instructions.The column was washed with 8 column volumes (CV) of water for Injection(WFI) and then charged using 3 CV of copper charging solution (5 mg/mLCuSO₄) using the flow rates recommended by the manufacturer. Followed bywashing with equilibration buffer (10 mM sodium phosphate/0.5 MNaCl/0.1% Triton X-100, pH 7.0) for 15 CV. The column was thenpre-washed with 25 CV of elution buffer (10 mM sodium phosphate/0.5 MNaCl/100 mM glycine/0.1% Triton X-100, pH 7.0) followed by 14 CV ofequilibration buffer (10 mM sodium phosphate/0.5 M NaCl/0.1% TritonX-100, pH 7.0) to remove any residual copper not tightly bound to theresin. The column was then loaded with 15 mg UspA2 diluted to 0.1 mg/mLin equilibration buffer (10 mM sodium phosphate/0.5 M NaCl/0.1% TritonX-100, pH 7.0). After loading, the column was washed again with 3 CV ofequilibration buffer (10 mM sodium phosphate/0.5 M NaCl/0.1% TritonX-100, pH 7.0). UspA2 was eluted in 10 mM sodium phosphate/0.5 MNaCl/0.1% Triton X-100, pH 7.0 for 10 CV over a linear gradient from0-100 mM glycine in order to determine the optimal concentration ofglycine for elution of the protein. SDS-PAGE and LOS Western Blotanalysis were performed on samples of the flow through during loading,washes after loading and fractions taken during elution of the protein.Those fractions containing UspA2 were pooled. The UspA2 fraction poolwas concentrated and diafiltered 5 fold over a 50 cm² 100K molecularweight cut-off (MWCO) regenerated cellulose Millipore membrane against10 mM sodium phosphate/100 mM glycine/0.025% Triton X-100, pH 7.0 toreduce residual copper. This was followed by 5 fold diafiltrationagainst PBS containing 0.025% Triton X-100 (10 mM sodium phosphate/150mM NaCl/0.025% Triton X-100, pH 7.0). The final diafiltered retentatewas then filtered through a 0.2 um Millipak 20.

The final batch concentrate was analyzed for total protein, SDS-PAGE,LAL and residual copper. The IMAC column reduced the endotoxin from 21.6EU/ug to <0.1 EU/ug based on LAL analysis (Table 1). The proteinrecovery was 83% based on BCA results after 0.2 um filtration. Residualcopper analysis indicated that there was 30 ppb copper remaining afterultrafiltration and 0.2 um filtration. SDS-PAGE analysis indicated thatUspA2 bound to the resin, as there was no protein detected in the columnflow through during loading or in the washes with equilibration bufferafter loading. UspA2 eluted off of the column between 20-50 mM glycinein 10 mM NaPO4/0.5 M NaCL/0.1% Triton X-100. LOS Western Blot analysisshowed that the endotoxin (LOS) did not bind to the resin, but came offduring loading and within 4 column volumes of washing in equilibrationbuffer after loading. The amount of endotoxin in the UspA2 containingfractions was less than 1 ng as determined by LOS Western Blot analysis.These fractions were pooled before proceeding to the next purificationstep in the process, which is ultrafiltration.

TABLE 1 Small Scale Chromatography SDS-PAGE Total Residual EstimatedProtein Copper Process Step Total Protein (BCA) LAL (ug/L) UspA2 Loadedonto 15 mg   15 mg 21.6 EU/ug ND IMAC Column UspA2 IMAC 11 mg   20 mg ND4594 ug/L Fraction Pool (4.6 ppm) UspA2 after 11 mg 17.8 mg ND NDUltrafiltration (contained a precipitate) UspA2 After 11 mg 12.5 mg <0.1EU/ug  30 ug/L Ultrafiltration/ (30 ppb)# 0.2 um filt. (precipitate wasremoved by 0.2 um filtration) #The specification for copper in U.S.drinking water is <1.3 ppm. ND = not detected at the lower limit ofdetection, which is <0.05 EU/ug for LAL and <2 ppb for copper.

Example 4 Scale up of IMAC Chromatography

An 1113 mL (9 cm×17.5 cm) column was packed using Pharmacia ChelatingSepharose Fast™ flow resin according to the manufacturer instructions.The column was charged using 2 CV of copper charging solution (5 mg/mLCuSO4). Followed by washing with 4 CV of WFI followed by equilibrationbuffer (10 mM sodium phosphate/0.5 M NaCl/0.1% Triton X-100, pH 6.8).The column was then pre-washed with 20 CV of elution buffer (10 mMsodium phosphate/0.5 M NaCl/100 mM glycine/0.1% Triton X-100, pH 7.0)followed by 10 additional column volumes of equilibration buffer (10 mMsodium phosphate/0.5 M NaCl/0.1% Triton X-100, pH 6.8) to remove anyresidual copper not tightly bound to the resin. The column was thenloaded with 99 mg UspA2 diluted to 0.1 mg/mL in equilibration buffer (10mM sodium phosphate/0.5 M NaCl/0.1% Triton X-100, pH 6.8). Afterloading, the column was washed again with 10 CV of equilibration buffer(10 mM sodium phosphate/0.5 M NaCl/0.1% Triton X-100, pH 6.8). A stepgradient was performed in 10 mM sodium phosphate/0.5M NaCl/100 mMglycine/0.1% Triton X-100, pH 6.8 for 15 CV to elute UspA2. SDS-PAGE andLOS Western Blot analysis were performed on samples of the flow throughduring loading, washes after loading and fractions taken during elutionof the protein. Those fractions containing UspA2 were pooled. The UspA2fraction pool was concentrated and diafiltered 13.5 fold over a 50 cm²100K regenerated cellulose Millipore membrane against PBS (10 mM sodiumphosphate/150 mM NaCl, pH 7.0). The final diafiltered retentate was thenfiltered through a 0.2 μm Millipak 20. The final batch concentrate wasanalyzed for total protein, SDS-PAGE, LAL, pyrogenicity and residualcopper.

SDS-PAGE analysis of column flow through indicated that UspA2 bound tothe resin as in Example 2 and there was no protein detected in thecolumn load or column washes in equilibration buffer after loading.UspA2 eluted after 1.5 column volumes of elution buffer and LOS WesternBlot analysis indicated that the endotoxin (LOS) did not bind to theresin but came off during loading and within 6 column volumes of washingin equilibration buffer after loading. LOS Western Blot analysisindicated that there was <1 ng LOS present in the fractions containingUspA2. These fractions were pooled (fractions 6 through 16) andconcentrated on a 100K regenerated cellulose filter. After concentrationby ultrafiltration, protein recovery was 76% by BCA analysis (Table 2).Twenty milligrams of the concentrated retentate was then diafilteredagainst 10 mM NaPO4/150 mM NaCl pH 7.2. Triton X-100 was not added tothe diafiltration buffer in order to minimize build-up of the detergenton the ultrafiltration membrane. Recovery after diafiltration was 100%,based on BCA analysis. There was no precipitation observed duringconcentration or diafiltration. Endotoxin was reduced from 21.6 EU/ug to<0.1 EU/ug. Residual copper analysis after ultrafiltration was 80 ppb.

TABLE 2 Scaled-up Chromatography SDS-PAGE Total Estimated ProteinResidual Process Step Total Protein BCA LAL Copper UspA2 Loaded 99 mg  99 mg 21.6 EU/ug ND onto IMAC Column UspA2 IMAC 90 mg   98 mg ND  11ppm Fraction Pool UspA2 after 90 mg 75.8 mg ND 6.4 ppm Concentrationbefore Diafiltration UspA2 After 19 mg   20 mg* <0.1 EU/ug   39 ppb^(#)Ultrafiltration/ 0.2 um filt. UspA2 After 60 mg   53 mg* <0.1 EU/ug   80ppb^(#) Ultrafiltration/ 0.2 um filt. *20 mg of the concentratedretentate was initially processed by diafiltration against 10 mM sodiumphosphate/150 mM NaCl pH 7.2. The remainder (53 mg) was diafiltered as aseparate aliquot to minimize loss in the event of diafiltration failure.#The specification for copper in U.S. drinking water is <1.3 ppm. ND =not detected at the lower limit of detection, which is <0.05 EU/ug forLAL and <2 ppb for copper.

Example 5 Chromatography Method Used for Determination of Resin Capacityfor UspA2

A 10 mL (1.6 cm×5 cm) column was packed with Pharmacia ChelatingSepharose Fast flow resin according to the manufacturer's instructions.The column was washed with a minimum of 8 CV of WFI, followed by aminimum of 3 CV of copper charging solution (5 mg/mL CuSO₄). The columnwas then washed with 20 additional WFI followed by >10 CV of elutionbuffer (10 mM sodium phosphate/0.5 M NaCL/100 mM glycine/0.1% TritonX-100, pH 7.0). After equilibrating the column with a minimum of 10 CVequilibration buffer (10 mM sodium phosphate/0.5 M NaCl/0.1% TritonX-100, pH 7.0), the column was loaded with different proteinconcentrations per milliliter of resin ranging from 1 mg/mL, 0.5 mg/mL,0.3 mg/mL. The protein was washed onto the column with a minimum of 10CV of equilibration buffer (10 mM sodium phosphate/0.5 M NaCl/0.1%Triton X-100, pH 7.0). Then eluted with a minimum of 10 CV elutionbuffer (10 mM sodium phosphate/0.5 M NaCL/100 mM glycine/0.1% TritonX-100, pH 7.0. SDS-PAGE was performed on samples of the flow throughduring loading, washes after loading and fractions taken during elutionof the protein.

SDS-Page analysis indicated that loading of the column at 1 mgUspA2/mLof resin and at 0.5 mg UspA2/mL of resin resulted in the flow through ofthe protein in the column during loading and the post-load wash withequilibration buffer (data not shown). This was not observed when thecolumn was loaded at 0.3 mg UspA2/mL resin. This suggests that using thebuffer conditions described in Example 4, the binding capacity of theresin for this protein is approximately 0.3-0.5 mg UspA2/mL of resin.

Example 6 Chromatography Method Used for Determining Elution Conditionsin Buffer Prepared at 0.04% Triton X-100 Concentration

An additional experiment was done in the same manner as described abovefor determination of resin capacity at 0.3 mg UspA2/mL of resin, exceptthe protein was eluted using an elution buffer containing 0.04% TritonX-100 instead of the elution buffer containing 0.1% Triton X-100. Thepurpose of this experiment was to determine if the protein and LOS woulddisplay the same properties if the Triton X-100 concentration waslowered. SDS-PAGE and LOS Western Blot analysis were performed onsamples of the flow through during loading, washes after loading andfractions taken during elution of the protein.

Reducing the Triton X-100 concentration from 0.1% to 0.04% appeared tohave no effect on the elution of UspA2 off of the column. LOS WesternBlot analysis indicated, as in previous experiments, that when thecolumn was loaded at 0.3 mg UspA2/mL of resin, the endotoxin did notbind to the resin and washed through the column during loading andduring the post-load washes with equilibration buffer (data not shown).

REFERENCES

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1. An isolated and purified Gram negative bacteria protein obtained by a method for removing bacterial endotoxin from a Gram negative bacterial protein solution, wherein the method uses immobilized metal ion affinity chromatography (IMAC) comprising the steps of: (a) applying the Gram negative bacteria protein solution to an IMAC resin, wherein the resin is equilibrated in a buffer; (b) eluting the endotoxin with the buffer of step (a), wherein the protein remains bound to the resin; (c) eluting the protein in an elution buffer comprising glycine; and (d) collecting the eluted protein, wherein the concentration of the endotoxin in the protein solution is effectively reduced by a factor of at least 200 and protein recovery is greater than at least 60%.
 2. The protein of claim 1, wherein the Gram negative bacteria is Moraxella catarrhalis.
 3. The protein of claim 2, wherein the protein is UspA2.
 4. The UspA2 protein of claim 3 comprising less than about 0.1 endotoxin units per ug of UspA2 protein.
 5. The protein of claim 1, wherein the glycine of step (c) is at a concentration of at least 1 mM to at least about 500 mM.
 6. The protein of claim 1, wherein the buffer of step (a) is selected from the group consisting of sodium phosphate, sodium acetate, 1,4-piperazinebis-(ethanesulfonic acid) (PIPES), N-(2-acetamido)iminodiacetic acid (ADA) and N-(2-acetamido)-2-aminoethanesulfonic acid (ACES).
 7. The protein of claim 6, wherein the buffer of step (a) is sodium phosphate at a concentration of at least about 1 mM to about 50 mM and has a pH of about 6.5 to about 7.8, the buffer further comprising about 150 mM NaCl and 0.1% Triton-X.
 8. The protein of claim 7, wherein the sodium phosphate has a concentration of about 10 mM and a pH of 6.8.
 9. The protein of claim 1, wherein the buffer of step (a) further comprises NaCl at a concentration of at least about 100 mM to about 500 mM.
 10. The protein of claim 1, wherein the elution buffer of step (c) is selected from the group consisting of sodium phosphate, sodium acetate, imidazole, histidine, ammonium chloride, PIPES, ADA and ACES.
 11. The protein of claim 10, wherein the elution buffer of step (c) is sodium phosphate at a concentration of at least about 1 mM to about 50 mM and has a pH of about 6.5 to about 7.8, the buffer further comprising about 150 mM NaCl, 0.04% Triton-X and 100 mM glycine.
 12. The protein of claim 11, wherein the sodium phosphate has a concentration of about 10 mM and a pH of 6.8.
 13. The protein of claim 1, wherein the elution buffer of step (c) further comprises NaCl at a concentration of at least about 100 mM to about 500 mM.
 14. The protein of claim 1, wherein the resin is charged with Cu²⁺, Ni²⁺, Co²⁺, Fe²⁺, Mn²⁺, Ti²⁺, Cd²⁺, Mg²⁺, Zn²⁺, or Fe³⁺.
 15. The protein of claim 14, wherein the resin is charged with 20 mM cupric sulfate.
 16. The protein of claim 1, wherein the buffer of step (a) and the elution buffer of step (c) each further comprise a mobile phase modifier selected from the group consisting of urea, isopropanol, ethanol, methanol, ethylene glycol and a detergent.
 17. The protein of claim 1, wherein the concentration of the endotoxin in the protein solution is effectively reduced by a factor of at least 2,500 and protein recovery is greater than at least 90%.
 18. The protein of claim 1, wherein the resin is washed prior to step (a) with at least 3 to 10 resin volumes of the buffer used in step (c) and then washed with 1 to 10 resin volumes of the buffer used in step (a), wherein the washes remove excess copper from the resin.
 19. The protein of claim 1, wherein the protein solution is diluted in a buffer comprising 10 mM sodium phosphate, 150 mM NaCl and 0.25% Triton-X at pH 6.9, before proceeding to step (a).
 20. The protein of claim 1, wherein the protein is further purified by ultrafiltration or filtration. 